Abstract: To study the adaptive evolution mechanism of Lactobacillus(L.)casei Zhang in the environment of antibiotics,yhaI gene knocked system was constructed and its resistant phenotype of the mutant was studied. The homologous arm sequences of yhaI gene were amplified by PCR respectively to construct the knockout vector with yhaI deletion. The mutant strain L. casei Zhang-G-1200-yhaI was constructed by using Cre/lox gene recombination system and double crossover L. casei Zhang-G-1200-yhaI::lox66-P32-cat-lox71.The antibiotic resistance phenotype of the mutants was studied by dilution method. The PCR and PCR products were verified by sequencing and the yhaI gene-deficient strains were obtained. The results of the resistant phenotype showed that when the gentamicin concentration were 8 and 16 μg/mL,the turbidity of the mutant strain became 1/2 of the wild strain. When the gentamicin concentration was 16 μg/mL,the viable count of the mutant strain became 7/10 of the wild strain. The successful construction of gene knockout system of L. casei Zhang provided a technical platform for the study of gene function validation of L. casei.